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S Gluck

Publications and source records attributed to S Gluck.

At least 37 records · Page 2Linked to original sources
PubMed1992

The osteoclast as a unicellular proton-transporting epithelium.

Osteoclasts dissolve bone mineral by the vectorial secretion of hydrogen ion at their osseous attachment site. To accomplish this, the osteoclast employs a vacuolar H+ ATPase that is polarized to the specialized proton-secreting plasma membrane domain, the ruffled border. Physiologically and biochemically, they resemble the specialized proton-secreting intercalated cells of the renal collecting duct, which also use a polarized vacuolar H+ ATPase to effect transepithelial hydrogen ion transport. Studies on the mechanism of hydrogen ion transport by the kidney may therefore provide insights into the control of acid secretion by the osteoclast.

Animals↗
PubMed1992

A plasma membrane proton ATPase in specialized cells of rat epididymis.

Acidification of the luminal fluid in the epididymis is believed to play an important role in sperm maturation. Previous studies have shown that specialized cells in the epithelium lining the epididymis contain high levels of carbonic anhydrase and that these cells have rod-shaped intramembraneous particles when examined by freeze fracture. Both of these features are characteristic of proton-transporting intercalated cells in the kidney collecting duct. We now show that apical cells in the head of the epididymis and clear cells in the body and tail of the epididymis express high levels of a vacuolar proton-pumping adenosinetriphosphatase on their apical plasma membranes and on intracellular vesicles. By analogy with kidney intercalated cells, these cell types may be specialized for acid secretion in the epididymis.

Animals↗
PubMed1992

cDNA sequence and tissue expression of bovine vacuolar H(+)-ATPase M(r) 70,000 subunit.

cDNA clones encoding the 70,000 relative molecular weight (M(r)) subunit of the bovine vacuolar proton-adenosine triphosphatase (H(+)-ATPase) were isolated, and a total length of 3.1 kb in overlapping clones from bovine kidney and brain libraries was sequenced. The cDNA contains a 1.9-kb coding region and yields a deduced protein sequence of 618 amino acids. The subunit sequence has 50% amino acid identity with the corresponding subunit from yeast, carrot, and Neurospora vacuolar H(+)-ATPases. The internal regions of the protein are most highly conserved, whereas the NH2- and COOH-terminals exhibit variability. mRNA levels of the M(r) 70,000 subunit were examined in multiple bovine tissues and were found to be expressed at highest levels in kidney medulla and cortex, at moderate levels in brain and adrenal gland, and at low levels in liver, muscle, and heart.

Adenosine Triphosphatases↗
PubMed1992

Expression of vacuolar H(+)-ATPase in mouse osteoclasts during in vitro differentiation.

Osteoclasts express high levels of a vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) on the ruffled membrane which they employ to dissolve bone mineral by acidifying their site of attachment on bone. The factors that control amplification of H(+)-ATPase during osteoclast differentiation are poorly understood. We examined the expression of vacuolar H(+)-ATPase in a cell culture system in which mouse spleen cells can be induced to differentiate into osteoclasts by coculture with a mouse bone marrow stromal cell line. We found that the coculture system produced active osteoclasts, identified as multinucleated cells with staining for tartrate-resistant acid phosphatase activity that formed genuine resorption pits in bone. These cells developed high levels of H(+)-ATPase expression in culture, and omission of dexamethasone or 1 alpha,25-dihydroxyvitamin D3 from the coculture system each partially suppressed the expression of H(+)-ATPase. The results demonstrate that the spleen and PA6 cell coculture system may be useful for investigating the factors that control the induction of H(+)-ATPase amplification that occurs during osteoclast differentiation.

Acid Phosphatase↗
PubMed1992

Polarized targeting of V-ATPase in kidney epithelial cells.

The membrane-associated V-ATPase that plays an important role in the regulation of acid-base balance by the kidney is a multisubunit enzyme that is densely packed into specialized membrane domains in intercalated cells. Intercalated cells can be separated into at least two subtypes, A-cells and B-cells, based on their morphological features, the distribution of V-ATPase, and the presence or absence of a basolateral chloride/bicarbonate anion exchanger (AE1) exclusively in B-cells. A-cells secrete protons into the tubule lumen, whereas B-cells secrete bicarbonate. The relative amounts of V-ATPase and AE1 in the plasma membranes of A- and B-cells are modulated under different acid-base conditions and provide a sensitive means by which urinary acidification can be controlled. The mechanisms governing the movement of acid-base transporting proteins between intracellular vesicles and the plasma membrane are under investigation. The microtubular apparatus of the cell is involved in maintaining both apical and basolateral polarity of the enzyme, and different isoforms of V-ATPase subunits may also be involved in the selective targeting of V-ATPase to different membrane domains.

Acid-Base Equilibrium↗
PubMed1992

V-ATPases of the plasma membrane.

V-ATPases reside in high densities on the plasma membrane in specialized types of insect and vertebrate cells. They provide unique biochemical and electrophysiological properties that allow them to function in energizing the plasma membrane in insects, and in cellular acid excretion in vertebrates.

Adenosine Triphosphatases↗
PubMed1991

Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells.

Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ ATPase in LLC-PK1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1 cells were allowed to internalize cytochrome c-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ ATPase in these compartments, we labeled LLC-PK1 cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ ATPase immunoprecipitated from both fractions with monoclonal anti-H+ ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.

Animals↗
PubMed1991

Colocalization of H(+)-ATPase and band 3 anion exchanger in rabbit collecting duct intercalated cells.

Two major types of intercalated cells (IC) have been previously defined in rabbit collecting duct: alpha-cells have a basolateral band 3-like anion exchanger and secrete H+, whereas beta-cells bind peanut agglutinin (PNA) apically and are believed to secrete HCO3-. To further define IC types, we double-labeled kidney sections with anti-H(+) -ATPase antibodies and with either an anti-band 3 antibody or PNA. We found four patterns of staining: 1) IC with apical H(+)-ATPase and basal band 3, a configuration consistent with ongoing H+ secretion, which prevailed in the inner stripe of outer medulla (OMCDi); 2) diffuse H(+)-ATPase labeling across the cell and basal band 3, which was most numerous in the outer stripe of outer medulla (OMCDo); 3) IC with "bright" apical peanut lectin, diffuse H(+)-ATPase, and no band 3, which was abundant in the cortical collecting duct (CCD) and probably represents HCO3(-)-secreting cells; and 4) "hybrid" cells with various staining combinations (e.g., apical lectin binding and apical H(+)-ATPase), which although they are uncommon, were seen in the CCD. Consistent with this immunocytochemical finding of hybrid cells, cell-sorting studies on isolated CCD IC showed that 6-18% of PNA-positive cells also stained positively for band 3. We conclude that 1) band 3-positive IC in the OMCD vary axially. Most OMCDi IC are probably active proton secretors, whereas up to one-half of OMCDo IC may be latent H+ secretors. 2) The diffuse H(+)-ATPase pattern in putative beta-cells differs from comparable results in the rat and is not consistent with a "reversed" alpha-cell. HCO3- secretion by beta-cells may be driven by an H+ extrusion mechanism other than the alpha-cell pump re-sorted to the basolateral membrane. 3) The possibility of hybrid cells that might combine alpha- and beta-cell transport proteins suggests a mechanism for functional reversal of collecting duct IC polarity.

Animals↗
PubMed1991

Cellular distribution of H(+)-ATPase following acute unilateral ureteral obstruction in rats.

Unilateral ureteral obstruction for 24 h produces an acidification defect in the rat kidney that closely resembles the human disorder. We examined the role of renal vacuolar H(+)-ATPase distribution and content in the generation of the postobstructive abnormality in distal hydrogen ion secretion. Rats were subjected to unilateral ureteral obstruction for 24 h, and the obstructed and contralateral kidneys were removed at 3 h, 5 days, and 10 days after release of the obstruction. The postobstructed and contralateral kidneys and kidneys from sham-operated rats were analyzed for intercalated cell number and subtype and for the cellular distribution of ATPase staining by means of a monoclonal antibody specific for the 31-kDa subunit of the vacuolar H(+)-ATPase. No change in the number or distribution of subtypes was detected in the cortex nor in the outer or inner stripe of the outer medulla. Immunoreactive H(+)-ATPase increased in both the cortex and medulla at 3 h after obstruction, and thereafter it declined to control values. The major morphological changes in H(+)-ATPase staining detected were an alteration in the intracellular distribution of the enzyme, which we refer to as discontinuity of (or "gaps" in) apical staining, and a decrease in the percent of intercalated cells showing a rim (or plasma membrane) staining pattern in the inner medulla. The changes observed may be a morphological representation of the physiological abnormalities underlying the postobstructive acidification defect.

Animals↗
PubMed1991

Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat.

Renal hydrogen ion excretion increases with chronic acid loads and decreases with alkali loads. We examined the mechanism of adaptation by analyzing vacuolar proton-translocating adenosine triphosphatase (H+ ATPase) 31-kD subunit protein and mRNA levels, and immunocytochemical distribution in kidneys from rats subjected to acid or alkali loads for 1, 3, 5, 7, and 14 d. Acid- and alkali-loaded rats exhibited adaptive responses in acid excretion, but showed no significant changes in H+ ATPase protein or mRNA levels in either cortex or medulla. In contrast, there were profound adaptive changes in the immunocytochemical distribution of H+ ATPase in collecting duct intercalated cells. In the medulla, H+ ATPase staining in acid-loaded rats shifted from cytoplasmic vesicles to plasma membrane, whereas in alkali-loaded rats, cytoplasmic vesicle staining was enhanced, and staining of plasma membrane disappeared. In the cortical collecting tubule, acid loading increased the number of intercalated cells showing enhanced apical H+ ATPase staining and decreased the number of cells with basolateral or poorly polarized apical staining. The results indicate that both medulla and cortex participate in the adaptive response to acid and alkali loading by changing the steady-state distribution of H+ ATPase, employing mechanisms that do not necessitate postulating interconversion of intercalated cells with opposing polarities.

Acid-Base Equilibrium↗
PubMed1991

Colchicine-induced redistribution of proton pumps in kidney epithelial cells.

In kidney medulla, intercalated cells (ICs) all have an apical proton-pumping ATPase, but in the cortex, ICs with apical (type A cells) and basolateral (type B cells) proton pumps coexist. Proton pumps in proximal-tubule epithelial cells are located in a band of apical membrane at the base of the brush border. To determine the effect of microtubule disruption on proton-pump polarity in these cell types, we immunolocalized proton pumps in normal and colchicine-treated rat kidneys. In addition, NEM-sensitive ATPase was measured in brush-border (BBMV) and basolateral membrane vesicles from kidney cortex. Rats were injected with colchicine, and kidneys were fixed 4-24 h later. In both cortical and medullary IC, proton pumps were localized on vesicles that were scattered throughout the cell. A- and B-type ICs were no longer distinguishable on the basis of proton-pump polarity. In the medulla, no concentration of proton pumps at the basolateral pole of ICs was detectable. In proximal tubules, pumps were also scattered on vesicles throughout the cytoplasm. NEM-sensitive ATPase was reduced by over 60% in BBMV, and was also reduced in basolateral membranes, indicating no increased insertion of NEM-sensitive ATPase into basolateral membranes after colchicine treatment. We conclude that a change in the polymerization state of microtubules is not sufficient to account for the differential targetting of proton pumps in subpopulations of intercalated cells.

Adenosine Triphosphatases↗
PubMed1990

Isolation and properties of bovine kidney brush border vacuolar H(+)-ATPase. A proton pump with enzymatic and structural differences from kidney microsomal H(+)-ATPase.

Vacuolar H(+)-ATPase was isolated from highly purified bovine kidney brush border, using a previously described immunoaffinity method. The affinity purified enzyme had reconstitutively active ATP-induced acidification that was inhibited by N-ethylmaleimide. The brush border H(+)-ATPase had a single pH optimum of 7.3, and a single Km for ATP of 360 microM. The enzyme showed no lipid activation; it had a substrate preference of ATP greater than ITP greater than UTP greater than GTP much greater than CTP, with an ATP:GTP selectivity of 1.69. The brush border H(+)-ATPase required no monovalent anion or cation for activity and was inhibited by the oxyanions NO3(-1) much greater than SO4(-2); sulfite stimulated activity at low concentrations and inhibited at higher concentrations. The inhibition produced by nitrate could not be attributed to dissociation of subunits from the enzyme. The divalent or trivalent cation preference was Mn+2 much greater than Mg+2 much greater than Co+2 greater than Al+3 greater than Ca+2 much greater than Ba+2,Sr+2; 1 mM Zn+2 inhibited the enzyme completely, but Cu+2 inhibited only 49% of activity at concentrations up to 5 mM. Sodium dodecyl sulfate-polyacrylamide gels of the brush border H(+)-ATPase showed subunits at Mr 70,000, a doublet at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000. On two-dimensional gels, the pl value for the Mr 70,000 subunit was 6.3, for the Mr 56,000 was 6.4, and for the Mr 31,000 was 7.5-8.5, and microheterogeneity was observed in the Mr 56,000 and 31,000 subunits. A comparison of kidney cortex brush border H(+)-ATPase with kidney cortex microsomal H(+)-ATPase revealed differences in pH optimum, Km for ATP, lipid dependence, substrate preference, divalent ion preference, copper sensitivity, and in microheterogeneity of the Mr 56,000 and 31,000 subunits, providing evidence that different functional and structural classes of vacuolar H(+)-ATPase are segregated to specific membrane compartments.

Adenosine Triphosphate↗
PubMed1990

Sequential expression of phenotype markers for osteoclasts during differentiation of precursors for multinucleated cells formed in long-term human marrow cultures.

Long-term human marrow cultures form multi-nucleated cells (MNC) which express the osteoclast phenotype. Mononuclear precursors for these MNC can be identified and highly enriched. We tested early (bipotent) and late (unipotent) precursors of these MNC for expression of several osteoclast differentiation markers: 1) the osteoclast vitronectin receptor, identified by the 23c6 monoclonal antibody, 2) the vacuolar-type proton pump, identified by the E11 monoclonal antibody, and 3) the calcitonin (CT) receptor, by autoradiography with 125I-labeled salmon calcitonin. We wished to determine if the proton pump was expressed in cells in long term marrow cultures and if its expression correlated with expression of the CT receptor and the vitronectin receptor. About 30% of early precursor cells reacted with the 23c6 monoclonal antibody, but none expressed CT receptors or showed amplified proton pump expression. The CT receptor and amplified proton pump expression were detected first on the late precursor, a stage in which every cell reacted strongly with the 23c6 monoclonal antibody. Over 80% of MNC formed from these late precursors expressed abundant CT receptors, and all MNC expressed the vitronectin receptor and showed amplified proton pump expression. In contrast, macrophage polykaryons and mononuclear macrophages formed in vitro failed to express the osteoclast vitronectin and CT receptors and expressed the proton pump at levels indistinguishable from those of surrounding cells.

Biomarkers↗
PubMed1989

Osteoclastic bone resorption by a polarized vacuolar proton pump.

Bone resorption depends on the formation, by osteoclasts, of an acidic extracellular compartment wherein matrix is degraded. The mechanism by which osteoclasts transport protons into that resorptive microenvironment was identified by means of adenosine triphosphate-dependent weak base accumulation in isolated osteoclast membrane vesicles, which exhibited substrate and inhibition properties characteristic of the vacuolar, electrogenic H+-transporting adenosine triphosphatase (H+-ATPase). Identify of the proton pump was confirmed by immunoblot of osteoclast membrane proteins probed with antibody to vacuolar H+-ATPase isolated from bovine kidney. The osteoclast's H+-ATPase was immunocytochemically localized to the cell-bone attachment site. Immunoelectron microscopy showed that the H+-ATPase was present in the ruffled membrane, the resorptive organ of the cell.

Animals↗
PubMed1989

Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase.

The cellular distributions of the kidney form of the erythrocyte band 3 chloride/bicarbonate exchanger and the kidney vacuolar H+-transporting ATPase were examined in rat kidney collecting duct by immunocytochemical staining of adjacent semithin sections. Polyclonal anti-peptide antibodies directed against two regions of murine erythroid band 3 gave a pattern of basolateral labeling similar to that seen with antibodies directed against the entire protein. In the medullary collecting duct almost all intercalated cells expressed basolateral membrane band 3 and displayed apical membrane H+-ATPase. In the cortical collecting duct and the connecting segment, band 3 labeling was restricted to a subpopulation of intercalcated cells. In the cortical collecting duct 46% of intercalated cells had apical H+-ATPase and basolateral band 3. Cells that had either basolateral or diffuse cytoplasmic staining for H+-ATPase were all band 3-negative and accounted for 53% of the intercalated cells. In addition, occasional intercalated cells with apical H+-ATPase appeared to lack basolateral band 3. These results demonstrate the coexpression of H+-ATPase and band 3 in opposite plasma membrane domains of a subpopulation of intercalated cells that are probably the acid-excreting (type A) cells. All other intercalated cells lacked immunoreactive band 3 and probably include the bicarbonate-excreting (type B) cells.

Animals↗
PubMed1988

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations.

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.

Animals↗
PubMed1988

Isolation and sequence of a cDNA clone encoding the 31-kDa subunit of bovine kidney vacuolar H+-ATPase.

We have isolated a cDNA encoding the 31-kDa subunit of the bovine kidney vacuolar H+-ATPase. The composite sequence contains 1219 base pairs, which includes the entire 678-base-pair coding region. A lysine-rich sequence previously found in the Na+, K+-ATPase alpha subunit and the H+,K+-ATPase was identified in the 31-kDa subunit. An RNA blot and an immunoblot demonstrated variable 31-kDa subunit expression and immunoreactivity in different tissues; the highest levels were observed in kidney medulla and brain with both types of analysis. The isolation of a cDNA for the 31-kDa subunit is an important step in understanding this subunit's role in the function and regulation of the vacuolar H+-ATPase.

Amino Acid Sequence↗